家蚕整合素β2的表达、纯化及其免疫功能

doi:10.3864/j.issn.0578-1752.2019.01.016

摘要/Abstract

摘要：

【目的】分析家蚕（Bombyx mori）整合素β2的序列和结构特征及其在家蚕感染病原菌后血细胞中的表达变化,检测其重组蛋白对不同病原相关模式分子识别和病原菌的凝聚作用,为进一步探究家蚕整合素β2的蛋白功能打下基础。【方法】利用生物信息学对整合素β2的序列和结构特征进行分析,利用实时荧光定量PCR检测家蚕分别注射大肠杆菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus）后整合素β2在血细胞中的表达变化。通过PCR技术扩增获得整合素β2完整的胞外域片段,构建至pET22b原核表达载体后转化至E.coli Rosetta(DE3)表达菌株。经IPTG诱导获得重组蛋白,利用Ni-NTA亲和层析得到纯化的体外重组蛋白,利用SDS-PAGE和Western blot对纯化获得的体外重组蛋白的纯度和质量进行检测。利用ELISA检测重组蛋白与两种不同病原相关分子模式LPS和PGN的结合情况,运用Western blot检测重组蛋白与不同病原微生物的结合情况,通过凝集试验检测重组蛋白对金黄色葡萄球菌的凝集能力,最后在个体水平探索重组蛋白在体内细菌清理中的作用。【结果】家蚕整合素β2具有典型的β整合素亚基保守结构特征,即由一个较长的胞外域、一个单次跨膜结构域和一个较短的胞内域构成。家蚕整合素β2具有金属离子结合位点MIDAS、EGF结构域、半胱氨酸重复基序和NPxY等整合素典型的结构特征。实时荧光定量PCR检测结果表明家蚕在受到细菌感染后,整合素β2的表达发生显著变化。通过原核表达和蛋白纯化获得纯度较高的重组蛋白,SDS-PAGE和Western blot检测结果表明纯化的重组蛋白纯度较高,可以用于后续试验。ELISA试验表明重组蛋白对LPS和PGN等病原相关分子模式具有较强的结合能力。细菌结合试验结果显示重组蛋白能够结合多种细菌,但与革兰氏阳性菌的结合能力要高于革兰氏阴性菌。细菌凝聚试验结果显示重组蛋白在Ca ²⁺的存在下对金黄色葡萄球菌具有较强的凝聚作用。细菌清除试验证实重组蛋白可以有效促进机体对外源入侵细菌的清理作用。【结果】整合素β2具有典型的整合素β亚基的结构特征,可能具有识别病原相关分子模式,如LPS和PGN等的能力,通过与细菌的直接结合而实现对入侵病原微生物的凝聚作用,从而增强有机体的免疫能力,推测在家蚕免疫反应中发挥重要作用。

关键词: 家蚕, 整合素β2;, 细菌感染, 原核表达, 细菌凝聚

Abstract:

【Objective】The objective of this study is to analyze the gene sequence and structural characteristics of integrin β2 in silkworm (Bombyx mori), and its expression profile in hemocytes following the larval exposure to different bacterial pathogens, investigate the binding and agglutination properties of the recombinant integrin β2 protein to various pathogen-associated molecular patterns (PAMPs) and bacteria, which will lay a foundation for further exploring the protein function of integrin β2 in B. mori. 【Method】Bioinformatics tools were used to determine the sequence and structural characteristics of integrin β2, and real-time fluorescent quantitative PCR (RT-qPCR) assay was executed to evaluate expression profile in hemocytes after microbial (Escherichia coli and Staphylococcus aureus) challenge. The cDNA fragment of integrin β2 was amplified using PCR, and the fragment containing the extracellular domain was inserted into a prokaryotic expression vector (pET22b). The insertion was confirmed in the recombinant plasmid and transformed into E. coli Rosetta (DE3), and then induced by IPTG to produce recombinant protein. The recombinant protein was purified using Ni-NTA affinity chromatography and analyzed by SDS-PAGE and Western blot. ELISA and Western blot were executed to determine the binding abilities of the recombinant protein to PAMPs (LPS and PGN) and different bacteria. Moreover, the agglutination ability and bacterial clearance assay were performed to understand the specific biological roles of integrin β2 in immunity.【Result】B. mori integrin β2 contains typical integrin β subunits, which comprises a long extracellular domain, a single transmembrane region and a short cytoplasmic tail. Further, it has several conserved motifs such as the MIDAS, EGF domain, Cys-repeat sequences and NPxY motifs. The RT-qPCR analysis showed that the integrin β2 expression varied significantly in hemocytes after infection with bacteria. High purity recombinant protein was obtained by prokaryotic expression and protein purification. The results of SDS-PAGE and Western blot showed that the purified recombinant protein was of high purity and could be used in subsequent tests. ELISA assay indicated that the purified recombinant integrin β2 protein had a strong binding ability to PAMPs (LPS and PGN). The results of bacterial binding test showed that the recombinant protein could bind many bacteria, but the binding ability with Gram-positive bacteria was higher than that with Gram-negative bacteria. The agglutination assay showed that the recombinant protein had strong agglutination effects on S. aureus in the presence of ca ²⁺. Further, bacterial clearance assay suggested that the recombinant protein could effectively promote the cleaning of exogenous invading bacteria from B. mori. 【Conclusion】The integrin β2 has a typical structure of the β integrin family, and it can recognize PAMPs (LPS and PGN) and enhance the aggregation of invading microbial pathogens by directly binding to them. Taken together, integrin β2 may play an important biological role in the bacterial immune response of B. mori.

Key words: Bombyx mori, integrin β2;, microbial challenge, prokaryotic expression, bacterial agglutination

张奎,李重阳,苏晶晶,谈娟,徐曼,崔红娟. 家蚕整合素β2的表达、纯化及其免疫功能[J]. 中国农业科学, 2019, 52(1): 181-190.

ZHANG Kui,LI ChongYang,SU JingJing,TAN Juan,XU Man,CUI HongJuan. Expression, Purification and Immunologic Function of Integrin β2 in the Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2019, 52(1): 181-190.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.01.016

https://www.chinaagrisci.com/CN/Y2019/V52/I1/181

图/表 7

图1

图2

图3

图4

图5

图6

图7

参考文献 35

[1]	TAKADA Y, YE X , SIMON S. The integrins. Genome Biology, 2007, 8(5): Article 215.
[2]	GIANCOTTI F G . A structural view of integrin activation and signaling. Developmental Cell, 2003,4(2):149-151. doi: 10.1016/S1534-5807(03)00034-0 pmid: 12586058
[3]	HUGHES A L . Evolution of the integrin α and β protein families. Journal of Molecular Evolution, 2001,52(1):63-72.
[4]	BANNO A, GINSBERG M H . The ins and outs of integrin signaling// LAFLAMME S E, KOWALCZYK A P. Cell Junctions: Adhesion, Development, and Disease. Wiley-VCH Verlag GmbH & Co. KGaA, 2008: 1-23.
[5]	HOOD J D, CHERESH D A . Role of integrins in cell invasion and migration. Nature Reviews Cancer, 2002,2(2):91-100. doi: 10.1038/nrc727 pmid: 12635172
[6]	HUTTENLOCHER A, HORWITZ A R . Integrins in cell migration. Cold Spring Harbor Perspectives in Biology, 2011,3(9):a005074.
[7]	BROOKS P C, MONTGOMERY A M, ROSENFELD M, REISFELD R A, HU T, KLIER G, CHERESH D A . Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 1994,79(7):1157-1164. doi: 10.1016/0092-8674(94)90007-8
[8]	MEREDITH J E, SCHWARTZ M A . Integrins, adhesion and apoptosis. Trends in Cell Biology, 1997,7(4):146-150. doi: 10.1016/S0962-8924(97)01002-7
[9]	GIANCOTTI F G, RUOSLAHTI E . Integrin signaling. Science, 1999,285(5430):1028-1032. doi: 10.1126/science.285.5430.1028
[10]	DESGROSELLIER J S, CHERESH D A . Integrins in cancer: biological implications and therapeutic opportunities. Nature Reviews Cancer, 2010,10(1):9-22. doi: 10.1038/nrc2748 pmid: 4383089
[11]	ZHANG K, XU M, SU J, YU S, SUN Z, LI Y, ZHANG W, HOU J, SHANG L, CUI H . Characterization and identification of the integrin family in silkworm, Bombyx mori. Gene, 2014,549(1):149-155. doi: 10.1016/j.gene.2014.07.060 pmid: 25064490
[12]	ZHANG K, TAN J, MAN X, SU J, HU R, CHEN Y, FAN X, RUI Y, CUI H . A novel granulocyte-specific α integrin is essential for cellular immunity in the silkworm Bombyx mori. Journal of Insect Physiology, 2014,71:61-67. doi: 10.1016/j.jinsphys.2014.10.007 pmid: 25450560
[13]	ZHANG K, TAN J, SU J, LIANG H, SHEN L, LI C, PAN G, YANG L, CUI H . Integrin β3 plays a novel role in innate immunity in silkworm, Bombyx mori. Developmental and Comparative Immunology, 2017,77:307-317.
[14]	孙亚兰 . 家蚕整合蛋白β基因的克隆及其表达分析[D]. 武汉: 华中师范大学, 2009.
	SUN Y L . cDNA cloning and gene expression of integrin β subunits from Bombyx mori[D]. Wuhan: Central China Normal University, 2009. ( in Chinese)
[15]	NAGAOSA K, OKADA R, NONAKA S, TAKEUCHI K, FUJITA Y, MIYASAKA T, MANAKA J, ANDO I, NAKANISHI Y . Integrin βν-mediated phagocytosis of apoptotic cells in Drosophila embryos. The Journal of Biological Chemistry, 2011,286(29):25770-25777.
[16]	XIE X, AULD V J . Integrins are necessary for the development and maintenance of the glial layers in the Drosophila peripheral nerve. Development, 2011,138(17):3813-3822. doi: 10.1242/dev.064816 pmid: 21828098
[17]	ZUSMAN S, GRINBLAT Y, YEE G, KAFATOS F C, HYNES R O . Analyses of PS integrin functions during Drosophila development. Development, 1993,118(3):737-750. doi: 10.1016/0165-3806(93)90096-S pmid: 8076515
[18]	BUNCH T A, GRANER M W, FESSLER L I, FESSLER J H, SCHNEIDER K D, KERSCHEN A, CHOY L P, BURGESS B W, BROWER D L . The PS2 integrin ligand tiggrin is required for proper muscle function in Drosophila. Development, 1998,125(9):1679-1689. doi: 10.1007/s004290050153 pmid: 9521906
[19]	BROWN N H . Cell-cell adhesion via the ECM: integrin genetics in fly and worm. Matrix Biology, 2000,19(3):191-201. doi: 10.1016/S0945-053X(00)00064-0 pmid: 10936444
[20]	SCHOTMAN H, KARHINEN L , RABOUILLE C. dGRASP- mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling. Developmental Cell, 2008,14(2):171-182.
[21]	LIN G, ZHANG X, REN J, PANG Z, WANG C, XU N, XI R . Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila. Developmental Biology, 2013,377(1):177-187. doi: 10.1016/j.ydbio.2013.01.032 pmid: 23410794
[22]	LAVINE M D, STRAND M R . Haemocytes from Pseudoplusia includens express multiple α and β integrin subunits. Insect Molecular Biology, 2003,12(5):441-452.
[23]	PECH L L, STRAND M R . Encapsulation of foreign targets by hemocytes of the moth Pseudoplusia includens( Lepidoptera: Noctuidae) involves an RGD-dependent cell adhesion mechanism. Journal of Insect Physiology, 1995,41(6):481-488. doi: 10.1016/0022-1910(94)00136-5
[24]	MAHAIRAKI V, LYCETT G , SIDÉN-KIAMOS I, SINDEN R E, LOUIS C. Close association of invading Plasmodium berghei and β integrin in the Anopheles gambiae midgut. Archives of Insect Biochemistry and Physiology, 2005,60(1):13-19.
[25]	MOITA L F, VRIEND G, MAHAIRAKI V, LOUIS C, KAFATOS F C . Integrins of Anopheles gambiae and a putative role of a new beta integrin, BINT2, in phagocytosis of E. coli. Insect Biochemistry and Molecular Biology, 2006,36(4):282-290.
[26]	MAMALI I, LAMPROU I, KARAGIANNIS F, KARAKANTZA M, LAMPROPOULOU M, MARMARAS V J . A β integrin subunit regulates bacterial phagocytosis in medfly haemocytes. Developmental and Comparative Immunology, 2009,33(7):858-866. doi: 10.1016/j.dci.2009.02.004 pmid: 19428487
[27]	HU J, ZHAO H, YU X, LIU J, WANG P, CHEN J, XU Q, ZHANG W . Integrin β1 subunit from Ostrinia furnacalis hemocytes: molecular characterization, expression, and effects on the spreading of plasmatocytes. Journal of Insect Physiology, 2010,56(12):1846-1856. doi: 10.1016/j.jinsphys.2010.08.001 pmid: 20708011
[28]	XU Q, YU X, LIU J, ZHAO H, WANG P, HU S, CHEN J, ZHANG W, HU J . Ostrinia furnacalis integrin β1 may be involved in polymerization of actin to modulate spreading and encapsulation of plasmatocytes. Developmental and Comparative Immunology, 2012,37(3/4):438-445. doi: 10.1016/j.dci.2012.02.003 pmid: 22343085
[29]	LEVIN D M, BREUER L N, ZHUANG S, ANDERSON S A, NARDI J B, KANOST M R . A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology, 2005,35(5):369-380. doi: 10.1016/j.ibmb.2005.01.003 pmid: 15804572
[30]	谈娟, 张奎, 徐曼, 陈思源, 崔红娟 . 家蚕整合素基因Bmintegrin αPS3的鉴定及亚细胞定位. 中国农业科学, 2013,46(22):4808-4815. doi: 10.3864/j.issn.0578-1752.2013.22.019
	TAN J, ZHANG K, XU M, CHEN S Y, CUI H Y . Identification and subcelluar localization of Bmintegrin αPS3 from silkworm( Bombyx mori). Scientia Agricultura Sinica, 2013,46(22):4808-4815. (in Chinese) doi: 10.3864/j.issn.0578-1752.2013.22.019
[31]	李重阳, 张奎, 申利, 赵羽卒, 潘光照, 徐曼, 苏晶晶, 崔红娟 . 家蚕整合素Bmintegrin β1基因的克隆及表达. 生物工程学报, 2017,33(12):1955-1967.
	LI C Y, ZHANG K, SHEN L, ZHAO Y Z, PAN G Z, XU M, SU J J, CUI H Y . Cloning and expression of Bmintegrin β1 in silkworm Bombyx mori. Chinese Journal of Biotechnology, 2017,33(12):1955-1967. (in Chinese)
[32]	ZHANG Y, WANG L, WANG L, WU N, ZHOU Z, SONG L . An integrin from shrimp Litopenaeus vannamei mediated microbial agglutination and cell proliferation. PLoS ONE, 2012,7(7):e40615. doi: 10.1371/journal.pone.0040615 pmid: 3392225
[33]	WANG Z, SHAO Y, LI C, LV Z, WANG H, ZHANG W, ZHAO X . A β-integrin from sea cucumber Apostichopus japonicus exhibits LPS binding activity and negatively regulates coelomocyte apoptosis. Fish and Shellfish Immunology, 2016,52:103-110.
[34]	HUANG Y, ZHAO L L, FENG J L, ZHU H X, HUANG X, REN Q, WEN W . A novel integrin function in innate immunity from Chinese mitten crab ( Eriocheir sinensis). Developmental and Comparative Immunology, 2015,52(2):155-165. doi: 10.1016/j.dci.2015.05.005 pmid: 26004499
[35]	JIA Z, ZHANG T, JIANG S, WANG M, CHENG Q, SUN M, WANG L, SONG L . An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity. Developmental and Comparative Immunology, 2015,53(1):253-264.